The free diffusion of macromolecules in tissue-engineered skeletal muscle subjected to large compression strains

Abstract

Pressure-related deep tissue injury (DTI) represents a severe pressure ulcer, which initiates in compressed muscle tissue overlying a bony prominence and progresses to more superficial tissues until penetrating the skin. Individual subjects with impaired motor and/or sensory capacities are at high risk of developing DTI. Impaired diffusion of critical metabolites in compressed muscle tissue may contribute to DTI, and impaired diffusion of tissue damage biomarkers may further impose a problem in developing early detection blood tests. We hypothesize that compression of muscle tissue between a bony prominence and a supporting surface locally influences the diffusion capacity of muscle. The objective of this study was therefore, to determine the effects of large compression strains on free diffusion in a tissue-engineered skeletal muscle model. Diffusion was measured with a range of fluorescently labeled dextran molecules (10, 20, 150 kDa) whose sizes were representative of both hormones and damage biomarkers. We used fluorescence recovery after photobleaching (FRAP) to compare diffusion coefficients (D) of the different dextrans between the uncompressed and compressed (48–60% strain) states. In a separate experiment, we simulated the effects of local partial muscle ischemia in vivo, by reducing the temperature of compressed specimens from 37 to 34 °C. Compared to the D in the uncompressed model system, values in the compressed state were significantly reduced by 47±22% (p<0.02). A 3 °C temperature decrease further reduced D in the compressed specimens by 10±6% (p<0.05). In vivo, the effects of large strains and ischemia are likely to be summative, and hence, the present findings suggest an important role of impaired diffusion in the etiology of DTI, and should also be considered when developing biochemical screening methods for early detection of DTI.

Introduction

Pressure-related deep tissue injury (DTI) represents a severe pressure ulcer, which initiates in muscle tissue overlying a bony prominence (e.g. the ischial tuberosities) and progresses to more superficial tissues. It eventually penetrates the skin surface with the potential of causing osteomyelitis, sepsis, renal failure, myocardial infarction and ultimate fatality (Agam and Gefen, 2007). Individual subjects with impaired motor and/or sensory capacities are particularly at risk of developing DTI, which is difficult to diagnose in a timely manner. Accordingly, the American and European Pressure Ulcer Advisory Panels are encouraging basic research into the etiology of DTI (Ankrom et al., 2005). Although the precise events leading to DTI are still poorly understood, a list of factors, all related to prolonged mechanical loading of muscle, have been suggested to initiate and/or accelerate tissue damage. These include ischemia (particularly hypoxia and pH decrease), metabolic waste product accumulation, ischemia–reperfusion cycles, obstruction/occlusion of lymphatic vessels, structural damage to cells, and rigor-mortis-like changes in mechanical properties of muscle (Bouten et al., 2003; Gefen et al., 2005).

Nevertheless, there is still surprisingly little published information related to those individual factors. For example, diffusion properties of muscle tissue play a critical role in the transport of metabolites (Swartz and Fleury, 2007); however, values for diffusion coefficients (D) of metabolites, particularly in loaded muscle, have not been adequately reported. The metabolic products of skeletal muscle range from molecular weights (MW) of less than 1 kDa (e.g. glucose) to many kDa (e.g. insulin), as summarized in Table 1. Of the few relevant papers in the literature, D of the water molecule (MW=18 Da) was examined under 60% compression strain in the isolated frog gastrocnemius muscle (Sekino et al., 2005). Using diffusion tensor MRI, they found that D of water decreased significantly by 11%. However, these data refer to non-viable tissue, tested at room temperature, and therefore, it is difficult to extrapolate to in vivo conditions. The effects of compression strains on D of larger molecules in muscle, at the scale of metabolic hormones or proteins released into the bloodstream when muscle damage occurs, and can thus be used as potential biomarkers of DTI in blood screening tests (Table 2), are unknown. It was observed that in compressed cartilage explants, though, 0.5–10 kDa molecules significantly decreased their diffusivity when strain increased from 10% to 50% (Evans and Quinn, 2005), and preliminary studies in tissue-engineered muscles stained with molecular markers at the 30 Da to 140 kDa range suggested a similar behavior (Gawlitta, 2007).

Accordingly, we hypothesize that mechanical compression influences the diffusion capacity of muscle tissue, and thus, causes a two-fold problem. (a) Diffusion of substances necessary for normal muscle metabolism may be hindered; depending on the MW of the substance, it may diffuse less across the extracellular space into or away from cells, or perhaps within cells. (b) If DTI occurs, diffusion of biomarkers indicating on occurrence of muscle damage into the blood circulation may be limited as long as the tissue is loaded, and this will interfere with early detection via blood tests (Gawlitta, 2007). Our objectives were therefore, to determine the effects of large compression strains on D of molecules with sizes representative of both hormones that regulate muscle metabolism and damage biomarkers, in tissue-engineered muscle constructs. We further investigated how D changes with a slight decrease in temperature (3 °C) to simulate localized temperature drops reported in ischemic skeletal muscle regions (Binzoni et al., 1990, Binzoni et al., 1998; Linder-Ganz and Gefen, 2007). Diffusion coefficients were determined using fluorescence recovery after photobleaching (FRAP), which allows reliable, site-specific measurements of D of fluorescently labeled molecule stains, following photobleaching of a geometrically defined small region with a high power laser (Seiffert and Oppermann, 2005).